1. The Field of the Invention
The present invention relates generally to optical microscopy. More particularly, the invention relates to a polarization-modulated tip enhanced optical microscope.
2. The Relevant Technology
Microscopy is the technical field of using microscopes to view samples or objects. There are three well-known branches of microscopy: optical, electron, and scanning probe. Optical and electron microscopy involve the diffraction, refraction, or reflection of electromagnetic radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image of the subject. This process may be carried out by wide field irradiation of the sample (for example, standard light microscopy and transmission electron microscopy) or by scanning of a fine beam over the sample (for example, confocal microscopy and scanning electron microscopy). Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest. The development of microscopy revolutionized biology and remains an essential tool in that science, along with many others.
Optical or light microscopy involves passing light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of the sample. More specifically, as light, such as a laser beam, is focused through a lens and onto a sample, the sample fluoresces. That is, as the light is directed onto the sample, the sample absorbs the light and emits light of a different wavelength. The resulting light can be detected directly by the eye, imaged onto a photographic plate, or captured digitally. The single lens with its attachments or the system of lenses and imaging equipment along with the appropriate lighting equipment, sample stage and support make up the basic light microscope.
While optical microscopy provides many benefits in imaging a sample, the resolution achieved with standard optical microscopy is limited. In particular, because standard optical microscopy uses light to image a sample, structures or features of the sample that are smaller than about half the wavelength of the light focused on the sample cannot be imaged. In biological samples, for example, the focused light is usually within the visible range of light, i.e., 500 to 800 nanometers. This means that sample structures or features that are smaller than 250 to 400 nanometers cannot be resolved or imaged using standard optical microscopy.
Many solutions have been designed to overcome these inherent limitations of the optical microscope, including tip-enhanced fluorescence microscopy (“TEFM”). TEFM is a type of apertureless near field scanning optical microscope (“ANSOM”) that utilizes fluorescence to generate an image. By aligning the sharp tip of a probe into the focus of a laser beam with axial polarization, enhanced fields are generated at the apex of the tip where the intensity of light is significantly greater than without the probe, analogous to a lightning rod. This field enhancement is tightly confined to the vicinity of the tip apex and has been shown to decay rapidly as r−6 with distance r from the tip apex. Therefore, light intensity measurements even a few nanometers away from the tip are lower than those at the tip and are about the same as if the tip were not present.
Once the high intensity region is created, it can be used to image nanometer scale features of a sample. When this technique is used to scan and image a sample, a background image is created. The background image is created by the light that is directed onto the sample away from the high intensity light region. This image is the same image that would be achieved by using a standard optical microscope without the addition of the probe tip, and thus the resolution is limited by diffraction. In addition to the background image, however, the high intensity region provides a greater detailed image of the sample within the high intensity region. As the sample is scanned, the more detailed image is superimposed on the background image, thereby providing a more detailed image of the complete sample.
The high intensity region enables information to be obtained about the distribution, structures, and features of the sample with much better resolution. In fact, the resolution is independent of the wavelength of the laser beam, and is only dependent on the size of the high intensity region. The size of the high intensity region is determined by the sharpness of the probe tip. In this manner, resolution can be achieved down to about 10 nanometers, thereby overcoming the diffraction limit discussed above by a factor of about 25.
The above identified modified optical microscope works well for imaging isolated molecules or particles on a surface when the molecules or particles are farther apart on average than the size of the laser beam. When they are close together and within the laser beam focus, the background signal goes up because each molecule or particle is fluorescing. However, because the region of higher intensity near the tip is so tightly localized, only one of the molecules or particles experiences the elevated field near the end of the tip. Thus, the more dense the sample is, the more molecules or particles there are within the laser focus, thereby reducing the image contrast because there is only one molecule that is being affected with the high intensity region near the tip, but there are many molecules within the laser that increase the background signal.
In response to problems associated with this poor contrast, methods for increasing the contrast between the background signal and that of the high intensity region have been developed. For example, in some applications, the probe tip is rapidly vibrated in and out of the region containing the sample to, in essence, rapidly modulate the detected signal. When the tip is moved close to the sample, the high intensity region at the tip's apex causes an increase in the detected signal. In contrast, when the tip is moved away from the sample, the signal returns to the background level. Any sample molecule within the high-intensity region will experience the higher intensity light, and the signal collected from that molecule will increase. However, the molecules outside of the higher intensity region, but within the area illuminated by the laser, will not experience the higher intensity light and the signal collected from them will, therefore, be the same as that resulting from the normal laser beam. In contrast, when the probe tip is moved away from the sample region, the high intensity region is removed from the single molecule, and the signal collected from that single molecule then returns to its normal level resulting from the laser beam.
In other words, oscillating or vibrating the tip quickly above the sample causes one molecule that is close to the tip to “blink” or fluctuate its fluorescence rate at the same rate that the tip is being oscillated. While one molecule within the high intensity region blinks, the signals given by molecules that are not close to the tip, but which are still within the laser focus, remain unchanged. Therefore, rather than detecting how much light is given off by a sample, the amount of “blinking” by each molecule is detected. In this manner the background signal can be suppressed while focusing on the one blinking molecule.
While oscillating the tip causes a molecule to blink, the blinking of the molecule is sensitive to the amplitude of the tip's oscillation. If the oscillation amplitude is too small then the fluctuation or blinking of the molecule is too small to detect or separate from the background signal. However, if the oscillation amplitude is too large, then the fluctuations or blinking will only occur for a brief period of time, that fraction of the oscillation period whereby the tip is close to the sample. Thus, there is an optimum oscillation amplitude for the tip. A limitation of the tip-oscillation method summarized above is that this optimum oscillation amplitude is rather large, (˜40 nm), which is sufficiently large as to possibly cause damage or unwanted perturbations to very soft samples, such as the thin membranes that envelope a cell or various organelles within the cell. This limits the utility of the technique when applied to a number of very important biological systems.
The subject matter claimed herein is not limited to embodiments that solve any disadvantages or that operate only in environments such as those described above. Rather, this background is only provided to illustrate one exemplary technology area where some embodiments described herein may be practiced.